Localization precision, STORM imaging and the tetrapod PSF

March 31, 2019

by Abhijit Marar

We were in San Francisco in February attending SPIE Photonics West. Our talk on incoherent holography was well received.

 

Localization Precision:

In order to measure the performance of the technique, we decided to calculate the localization precision by localizing 100 images of a single 100 nm red fluorescent microsphere (580/605). More information on localization precision can be found in Deschout, Hendrik, et al. "Precisely and accurately localizing single emitters in fluorescence microscopy." Nature methods 11.3 (2014): 253.  The results of the localization precision are shown below.

 

 

STORM imaging of neuron:

We are continuing our collaboration with Dr. Kamiyama from the Biology department to perform 2D- STORM imaging of the neuro-muscular junction. They have worked out the staining conditions for imaging the neuron which will be stained with AF 647 and AF 405 dye. He is still to figure out the conditions to express the fluorescent protein eos in the muscle. Below is a widefield (left) and STORM (right) image of a the growth cone stained with AF 647 and AF 405.

 

Understanding the tetrapod PSF:

We are also trying to compare incoherent holography with the tetrapod PSF which is another PSF engineering technique with the potential to produce high-resolution images across large axial ranges (20 µm). Further information about the tetrapod PSF can be found at Shechtman, Yoav, et al. "Precise three-dimensional scan-free multiple-particle tracking over large axial ranges with tetrapod point spread functions." Nano letters 15.6 (2015): 4194-4199. Below is an image showing the detrimental effect of overlapping emitters on the precision of the 10 µm tetrapod PSF.